Peroxynitrite is a reactive oxidant produced from nitric oxide (NO) and superoxide, which reacts with a variety of biomolecules including proteins, lipids and DNA. Peroxynitrite is produced by the body in response to a variety of toxicologically relevant molecules including environmental toxins. It is also produced by the body in response to environmental toxins, as well as in reperfusion injury and inflammation. Here we overview the multiple pathways of peroxynitrite cytotoxicity. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na(+)/K(+) ATP-ase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of peroxynitrite. In addition, peroxynitrite is a potent trigger of DNA strand breakage, with subsequent activation of the nuclear enzyme poly-ADP ribosyl synthetase or polymerase (PARP), with eventual severe energy depletion and necrosis of the cells. Studies conducted with peroxynitrite decomposition catalysts suggest that neutralization of peroxynitrite is of significant therapeutic benefit after exposure to various environmental toxins as well as in a variety of inflammatory and reperfusion disease conditions.

Learning in a constant environment, and adapting flexibly to a changing one, through changes in reinforcement contingencies or valence-free cues, depends on overlapping circuitry that interconnects the prefrontal cortex (PFC) with the striatum and is subject to several forms of neurochemical modulation. We present evidence from recent studies in animals employing electrophysiological, pharmacological and lesion techniques, and neuroimaging, neuropsychological and pharmacological investigations of healthy humans and clinical patients. Dopamine (DA) neurotransmission in the medial striatum and PFC is critical for basic reinforcement learning and the integration of negative feedback during reversal learning, whilst orbitofrontal 5-hydroxytryptamine (5-HT) likely mediates this type of low level flexibility, perhaps by reducing interference from salient stimuli. The role of prefrontal noradrenaline (NA) in higher order flexibility indexed through attentional set-shifting has recently received significant empirical support, and similar avenues appear promising in the field of task switching.

Diffuse axonal injury (DAI) is one of the most common and important pathologies resulting from the mechanical deformation of the brain during trauma. It has been hypothesized that calcium influx into axons plays a major role in the pathophysiology of DAI. However, there is little direct evidence to support this hypothesis, and mechanisms of potential calcium entry have not been explored. In the present study, we used an in vitro model of axonal stretch injury to evaluate the extent and modulation of calcium entry after trauma. Using a calcium-sensitive dye, we observed a dramatic increase in intra-axonal calcium levels immediately after injury. Axonal injury in a calcium-free extracellular solution resulted in no change in calcium concentration, suggesting an extracellular source for the increased post-traumatic calcium levels. We also found that the post-traumatic change in intra-axonal calcium was completely abolished by the application of the sodium channel blocker tetrodotoxin or by replacement of sodium with N-methyl-d-glucamine. In addition, application of the voltage-gated calcium channel (VGCC) blocker omega-conotoxin MVIIC attenuated the post-traumatic increase in calcium. Furthermore, blockade of the Na(+)-Ca(2+) exchanger with bepridil modestly reduced the calcium influx after injury. In contrast to previously proposed mechanisms of calcium entry after axonal trauma, we found no evidence of calcium entry through mechanically produced pores (mechanoporation). Rather, our results suggest that traumatic deformation of axons induces abnormal sodium influx through mechanically sensitive Na(+) channels, which subsequently triggers an increase in intra-axonal calcium via the opening of VGCCs and reversal of the Na(+)-Ca(2+) exchanger.

The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.

Recent advances in neurobiology have aided our understanding of attention-deficit hyperactivity disorder (ADHD). The higher-order association cortices in the temporal and parietal lobes and prefrontal cortex (PFC) interconnect to mediate aspects of attention. The parietal association cortices are important for orienting attentional resources in time/space, while the temporal association cortices analyse visual features critical for identifying objects/places. These posterior cortices are engaged by the salience of a stimulus (its physical characteristics such as movement and colour). Conversely, the PFC is critical for regulating attention based on relevance (i.e. its meaning). The PFC is important for screening distractions, sustaining attention and shifting/dividing attention in a task-appropriate manner. The PFC is critical for regulating behaviour/emotion, especially for inhibiting inappropriate emotions, impulses and habits. The PFC is needed for allocating/planning to achieve goals and organizing behaviour/thought. These regulatory abilities are often referred to as executive functions. In humans, the right hemisphere of the PFC is important for regulating distractions, inappropriate behaviour and emotional responses. Imaging studies of patients with ADHD indicate that these regions are underactive with weakened connections to other parts of the brain. The PFC regulates attention and behaviour through networks of interconnected pyramidal cells. These networks excite each other to store goals/rules to guide actions and are highly dependent on their neurochemical environment, as small changes in the catecholamines noradrenaline (NA) or dopamine (DA) can have marked effects on PFC function. NA and DA are released in the PFC according to our arousal state; too little (during fatigue or boredom) or too much (during stress) impairs PFC function. Optimal amounts are released when we are alert/interested. The beneficial effects of NA occur at postsynaptic alpha(2A)-receptors on the dendritic spines of PFC pyramidal cells. Stimulation of these receptors initiates a series of chemical events inside the cell. These chemical signals lead to the closing of special ion channels, thus strengthening the connectivity of network inputs to the cell. Conversely, the beneficial effects of moderate amounts of DA occur at D(1) receptors, which act by weakening irrelevant inputs to the cells on another set of spines. Genetic linkage studies of ADHD suggest that these catecholamine pathways may be altered in some families with ADHD, e.g. alterations in the enzyme that synthesizes NA (DA beta-hydroxylase) are associated with weakened PFC abilities. Pharmacological studies in animals indicate catecholamine actions in the PFC are highly relevant to ADHD. Blocking NA alpha(2A)-receptors in the PFC with yohimbine produces a profile similar to ADHD: locomotor hyperactivity, impulsivity and poor working memory. Conversely, drugs that enhance alpha(2)-receptor stimulation improve PFC function. Guanfacine directly stimulates postsynaptic alpha(2A)-receptors in the PFC and improves functioning, while methylphenidate and atomoxetine increase endogenous NA and DA levels and indirectly improve PFC function via alpha(2A)- and D(1) receptor actions. Methylphenidate and atomoxetine have more potent actions in the PFC than in subcortical structures, which may explain why proper administration of stimulant medications does not lead to abuse. Further understanding of the neurobiology of attention and impulse control will allow us to better tailor treatments for specific patient needs.

The roles of H+-Na+ and Na+-Ca2+ exchange in the depression of ventricular function were studied in the reperfused isolated ischemic rat heart. Zero-flow global ischemia was induced for either 15 or 30 minutes and was followed by 30 minutes of aerobic reperfusion. Intracellular Na+ (Na+i) and 45Ca2+ uptake were measured during ischemia and reperfusion. Accumulation of Na+i was modified by prior glycogen depletion and by treatment with amiloride, a H+-Na+ exchange inhibitor, or monensin, a Na+ ionophore. Na+i rose continuously during ischemia and rapidly during the first two minutes of reperfusion. The larger inhibitory effect of amiloride and preischemic glycogen depletion was on Na+i accumulation during reperfusion; this finding suggests that the uptake occurs by H+-Na+ exchange. Reduction of Na+i accumulation by glycogen depletion was associated with less lactate and, presumably, H+ production and accumulation during ischemia. The rapid increase in Na+i during early reperfusion may reflect the readjustment of the low intracellular pH resulting from ischemia. The level of Na+i at the end of ischemia and especially after two minutes of reperfusion were linearly correlated with 45Ca2+ uptake and depression of ventricular function during subsequent reperfusion. This highly significant correlation between Na+i and 45Ca2+ uptake when Na+i was varied by several independent procedures, including monensin, strongly suggests that reperfusion 45Ca2+ uptake occurs at least in part by Na+-Ca2+ exchange. The rate of 45Ca2+ uptake during reperfusion was linearly and highly significantly correlated with elevation of diastolic pressure, reduced developed pressure, and decreased recovery of ventricular function. The data strongly support a mechanism of ischemic cell damage that involves excessive production and accumulation of H+ during ischemia that exchanges for extracellular Na+ during ischemia and rapidly during the first few minutes of reperfusion. Increased Na+i then causes excessive 45Ca2+ uptake and depressed recovery of cellular functions with continued reperfusion. Increased levels of Na+i may be a major event that couples a decreased intracellular pH during ischemia to excessive 45Ca2+ uptake and depressed recovery of cellular function with reperfusion.

Cecropins, positively charged antibacterial peptides found in the cecropia moth, and synthetic peptide analogs form large time-variant and voltage-dependent ion channels in planar lipid membranes in the physiological range of concentration. Single-channel conductances of up to 2.5 nS (in 0.1 M NaCl) were observed, which suggests a channel diameter of 4 nm. Channels formed by the peptides cecropin AD and MP3 had a permeability ratio of Cl-/Na+ = 2:1 in 0.1 M NaCl. A comparative study of the three cecropins, cecropins A, B, and D, and of six synthetic analogs allowed determination of structural requirements for pore formation. Shorter amphipathic peptides did not form channels, although they adsorbed to the bilayer. A flexible segment between the N-terminal amphipathic region and the C-terminal more hydrophobic region of the peptide was required for the observation of a time-variant, voltage-dependent conductance. Cecropin AD was the most effective voltage-dependent pore-forming peptide and was also the most potent antibacterial peptide against several test organisms. A positive surface charge or cholesterol in the bilayer reduced the conductances caused by cecropin AD or MP3 by at least 5-fold. This behavior is consistent with the known insensitivity of eukaryotic cells to cecropins. Our observations suggest that the broad antibacterial activity of cecropins is due to formation of large pores in bacterial cell membranes.

The uptake of a sugar across the boundary membrane is a primary event in the nutrition of most cells, but the hydrophobic nature of the transport proteins involved makes them difficult to characterize. Their amino-acid sequences can, however, be determined by cloning and sequencing the corresponding gene (or complementary DNA). We have determined the sequences of the arabinose-H+ and xylose-H+ membrane transport proteins of Escherichia coli. They are homologous with each other and, unexpectedly, with the glucose transporters of human hepatoma and rat brain cells. All four proteins share similarities with the E. coli citrate transporter. Comparisons of their sequences and hydropathic profiles yield insights into their structure, functionally important residues and possible evolutionary relationships. There is little apparent homology with the lactose-H+ (LacY) or melibiose-Na+ (MelB) transport proteins of E. coli.

An inhibitory neurotransmitter in mature brain, gamma-aminobutyric acid (GABA) also appears to be excitatory early in development. The mechanisms underlying this shift are not well understood. In vitro studies have suggested that Na-K-Cl cotransport may have a role in modulating immature neuronal and oligodendrocyte responses to the neurotransmitter GABA. An in vivo developmental study would test this view. Therefore, we examined the expression of the BSC2 isoform of the Na-K-2Cl cotransporter in the postnatal developing rat brain. A comparison of sections from developing rat brains by in situ hybridization revealed a well-delineated temporal and spatial pattern of first increasing and then diminishing cotransporter expression. Na-K-2Cl mRNA expression in the cerebral cortex and hippocampus was highest in the first week of postnatal life and then diminished from postnatal day (PND) 14 to adult. Cotransporter signal in white-matter tracts of the cerebrum, cerebellum, peaked at PND 14. Expression was detected in cerebellar progenitor cells of the external granular layer, in internal granular layer cells at least as early as PND 7, and in Purkinje cells beginning at PND 14. Double-labeling immunofluorescence of brain sections with anti-BSC2 antibody and cell type-specific antibodies confirmed expression of the cotransporter gene product in neurons and oligodendrocytes in the white matter in a pattern similar to that determined by in situ hybridization. The temporal pattern of expression of the Na-K-2Cl cotransporter in the postnatal rat brain supports the hypothesis that the cotransporter is the mechanism of intracellular Cl- accumulation in immature neurons and oligodendrocytes.

ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of [2Fe-2S] centers prior to delivery to an apo target protein. The intermediate [2Fe-2S] ISU-bound cluster is formed by delivery of iron and sulfur to the apo ISU, with the latter delivered through an IscS-mediated reaction. The identity of the iron donor has thus far not been established. In this paper we demonstrate human frataxin to bind from six to seven iron ions. Iron binding to frataxin has been quantitated by iron-dependent fluorescence measurements [K(D)(Fe(3+)) approximately 11.7 microM; (K(D)(Fe(2+)) approximately 55.0 microM] and isothermal titration calorimetry (ITC) [K(D)(Fe(3+)) approximately 10.2 microM]. Enthalpies and entropies for ferric ion binding were determined from calorimetric measurements. Both fluorescence (K(D) 0.45 microM) and ITC measurements (K(D) 0.15 microM) demonstrate holo frataxin to form a complex with ISU with sub-micromolar binding affinities. Significantly, apo frataxin does not bind to ISU, suggesting an important role for iron in cross-linking the two proteins and/or stabilizing the structure of frataxin that is recognized by ISU. Holo frataxin is also shown to mediate the transfer of iron from holo frataxin to nucleation sites for [2Fe-2S] cluster formation on ISU. We have demonstrated elsewhere [J. Am. Chem. Soc. 2002, 124, 8774-8775] that this iron-bound form of ISU is viable for assembly of holo ISU, either by subsequent addition of sulfide or by NifS-mediated sulfur delivery. Provision of holo frataxin and inorganic sulfide is sufficient for cluster assembly in up to 70% yield. With NifS as a sulfur donor, yields in excess of 70% of holo ISU were obtained. Both UV-vis and CD spectroscopic characteristics were found to be consistent with those of previously characterized ISU proteins. The time course for cluster assembly was monitored from the 456 nm absorbance of holo ISU formed during the [2Fe-2S] cluster assembly reaction. A kinetic rate constant k(obs) approximately 0.075 min(-)(1) was determined with 100 microM ISU, 2.4 mM Na(2)S, and 40 microM holo frataxin in 50 mM Tris-HCl (pH 7.5) with 4.3 mM DTT. Similar rates were obtained for NifS-mediated sulfur delivery, consistent with iron release from frataxin as a rate-limiting step in the cluster assembly reaction.

The Na(+)-K(+) co-transporter HKT1, first isolated from wheat, mediates high-affinity K(+) uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar. AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na(+) uptake transporter in Xenopus laevis oocytes, and the presence of external K(+) did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na(+), which correlates to the large inward Na(+) currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K(+) uptake when cultured in K(+)-limiting medium. However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K(+) transporters. The rescue was associated with a less than 2-fold stimulation of K(+) uptake into K(+)-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na(+) and, to a small degree, K(+) transport in heterologous expression systems.

Exposure of HeLa cells to Na butyrate leads to an accumulation of multiacetylated forms of histones H3 and H4. Our studies of histone acetylation in HeLa S-3 cells show that 7 mM butyrate suppresses the deacetylation of histones without influencing the rate of radioactive acetate incorporation. An alteration in nucleosome structure in highly acetylated chromatin is indicated by an increased rate of DNA degradation by DNase I. A close association of acetylated histones with the DNase I-sensitive sequences is confirmed by the finding that histones remaining after limited DNase I digestion are depleted in the multiacetylated forms of histones H3 and H4. DNase I treatment has also been found to selectively release [3H]acetyl-labeled H3 and H4 from avian erythrocyte nuclei under conditions previously shown to preferentially degrade the globlin genes in erthyrocyte chromatin. Our results are consistent with the view that histone acetylation provides a key to the mechanism for altering chromatin structure at the nucleosomal level, and that this may explain the selective DNase I sensitivity of transcriptionally active DNA sequences in different cell types.

Electrically silent Na(+)-(K+)-Cl- transporter systems are present in a wide variety of cells and serve diverse physiological functions. In chloride secretory and absorbing epithelia, these cotransporters provide the chloride entry mechanism crucial for transcellular chloride transport. We have isolated cDNAs encoding the two major electroneutral sodium-chloride transporters present in the mammalian kidney, the bumetanide-sensitive Na(+)-K(+)-Cl- symporter and thiazide-sensitive Na(+)-Cl- cotransporter, and have characterized their functional activity in Xenopus laevis oocytes. Despite their differing sensitivities to bumetanide and thiazides and their different requirements for potassium, these approximately 115-kDa proteins share significant sequence similarity (approximately 60%) and exhibit a topology featuring 12 potential membrane-spanning helices flanked by long non-hydrophobic domains at the NH2 and COOH termini. Northern blot analysis and in situ hybridization indicate that these transporters are expressed predominantly in kidney with an intrarenal distribution consistent with their recognized functional localization. These proteins establish a new family of Na(+)-(K+)-Cl- cotransporters.

A Ca2+-channel blocker derived from funnel-web spider toxin (FTX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na+-dependent plateau potential. In the squid giant synapse, FTX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2+ current and of transmitter release. FTX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10- to 12-pS conductance in 80 mM Ba2+ and 5-8 pS in 100 mM Ca2+ with voltage-dependent open probabilities and kinetics. High Ba2+ concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic Ba2+. Both channels were blocked by FTX, Cd2+, and Co2+ but were not blocked by omega-conotoxin or dihydropyridines. These results suggest that one of the main Ca2+ conductances in mammalian neurons and in the squid preterminal represents the activation of a previously undefined class of Ca2+ channel. We propose that it be termed the "P" channel, as it was first described in Purkinje cells.

Transient receptor potential vanilloid 1 (TRPV1) is an ion channel that is gated by noxious heat, capsaicin and other diverse stimuli. It is a nonselective cation channel that prefers Ca2+ over Na+. These permeability characteristics, as in most channels, are widely presumed to be static. On the contrary, we found that activation of native or recombinant rat TRPV1 leads to time- and agonist concentration-dependent increases in relative permeability to large cations and changes in Ca2+ permeability. Using the substituted cysteine accessibility method, we saw that these changes were attributable to alterations in the TRPV1 selectivity filter. TRPV1 agonists showed different capabilities for evoking ionic selectivity changes. Furthermore, protein kinase C-dependent phosphorylation of Ser800 in the TRPV1 C terminus potentiated agonist-evoked ionic selectivity changes. Thus, the qualitative signaling properties of TRPV1 are dynamically modulated during channel activation, a process that probably shapes TRPV1 participation in pain, cytotoxicity and neurotransmitter release.

BACKGROUND

Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (Na(V)1.5) to this region.

OBJECTIVE

To establish the association of PKP2 with sodium channels and its role on action potential propagation.

RESULTS

Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with Na(V)1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes.

CONCLUSIONS

These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed.

Erythromelalgia is an autosomal dominant disorder characterized by burning pain in response to warm stimuli or moderate exercise. We describe a novel mutation in a family with erythromelalgia in SCN9A, the gene that encodes the Na(v)1.7 sodium channel. Na(v)1.7 produces threshold currents and is selectively expressed within sensory neurons including nociceptors. We demonstrate that this mutation, which produces a hyperpolarizing shift in activation and a depolarizing shift in steady-state inactivation, lowers thresholds for single action potentials and high frequency firing in dorsal root ganglion neurons. Erythromelalgia is the first inherited pain disorder in which it is possible to link a mutation with an abnormality in ion channel function and with altered firing of pain signalling neurons.

1. A study has been made of the cellular content and movement of Ca across the membrane of human red blood cells.2. The [Ca] in the cellular contents of fresh red cells is 4.09 x 10(-2) mM. The intracellular concentration of free ionic Ca ([Ca(2+)]) is considered to be less than this value and therefore less than extracellular [Ca(2+)] under normal conditions.3. Observation of unidirectional Ca fluxes with (45)Ca confirms previous reports of low permeability of the red cell membrane for Ca. After nearly 1 week of loading in the cold, intracellular (45)Ca content is 1.8% of extracellular (45)Ca content. Appearance in extracellular fluid of (45)Ca from coldloaded cells can be considered to arise from two compartments. Efflux of (45)Ca from the ;slower compartment' is accelerated by the addition of glucose.4. Starved red cells, incubated at 37 degrees C, after reversible haemolysis for loading with Ca and Mg-ATP, exhibit an outward net transport of Ca against an electrochemical gradient. The transport is associated with the appearance of inorganic phosphate (P(i)). Cells treated similarly, but without ATP show no transport and no appearance of P(i).5. During the initial phase of transport, 1.3 mole P(i) appear per mole Ca transported.6. The transport of Ca from ATP-loaded cells is highly temperature-dependent, with a Q(10) of 3.5.7. Cell membrane adenosine triphosphatase (ATPase) activity of reversibly haemolysed cells is stimulated only by intracellular, and not by extracellular Ca.8. Neither Ca transport in reversibly haemolysed cells, nor the Ca-Mg activated ATPase of isolated cell membranes is sensitive to Na, K, ouabain or oligomycin.9. Mg is not transported under the conditions which reveal Ca transport, but Mg appears to be necessary for Ca transport.10. Sr is transported from reversibly haemolysed Mg-ATP-loaded cells. Sr also can substitute for Ca, but not for Mg, in the activation of membrane ATPase.11. It is concluded that, in addition to a low passive permeability, an active extrusion mechanism for Ca exists in the human red cell membrane. This extrusion mechanism, in addition to a low passive membrane permeability for Ca, may represent the means by which intracellular Ca content is maintained at a low level. It is suggested that the Ca-Mg activated membrane ATPase and the active transport of Ca are two manifestations of the same process.

The sense of smell is highly evolved in mammals, allowing discrimination between a vast number of odorants, with detection thresholds as low as 10(-17) M (ref. 1). Although several features of mammalian olfactory transduction have been revealed by biochemical and molecular biological studies, the odorant-induced membrane current has remained elusive. In amphibians this current is mediated by cyclic-nucleotide-gated channels, which depolarize the cell by Na+ and Ca+ influx and consequent Cl- efflux through Ca(2+)-dependent Cl- channels. The Cl- current may be absent in mammals, however, because its proposed role is linked to the aquatic habitat of amphibians. Here we show that the transduction current in rat olfactory receptor cells is initiated by cyclic-nucleotide-gated channels. The Cl- current is also present and endows the transduction current with a steep sigmoidal dependence on cyclic AMP concentration in both rat and in an amphibian, indicating a new function for the Cl- channel: nonlinear amplification of the transduction signal, whereby suprathreshold responses are boosted relative to basal transduction noise.

Fluorescent indicators sensitive to cytosolic concentrations of free Na+ have been synthesized and characterized. They consist of a crown ether, 1,7-diaza-4,10,13-trioxacyclopentadecane, linked via its nitrogens to fluorophores bearing additional liganding centers. In the currently preferred dye, SBFI (short for sodium-binding benzofuran isophthalate), the fluorophores are benzofurans linked to isophthalate groups. Selectivities for Na+ over K+ of about 20 are observed, resulting in effective dissociation constants for Na+ of about 20 mM against a background of 120 mM K+. Increasing [Na+] increases the ratio of excitation efficiency at 330-345 nm to that at 370-390 nm with emission collected at 450-550 nm, so that ratio fluorometry and imaging can be performed with the same wavelengths as used with the well known Ca2+ indicator fura-2. If the macrocyclic ring is increased in size to a 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane, the chelators become selective for K+ over Na+.

Tight junctions (TJ) regulate paracellular ionic charge selectivity and conductance across epithelial tissues and cell lines. These properties vary among epithelia, and recent evidence implicates the claudins, a family of TJ transmembrane proteins, as important determinants of both characteristics. To test the hypothesis that each claudin contributes a characteristic charge discrimination to the TJ, we expressed claudins-2, -4, -11, and -15 in both cation-selective Madin-Darby canine kidney (MDCK) II cells and in anion-selective LLC-PK1 cells and examined changes in transepithelial electrical resistance (TER) and paracellular charge selectivity. Regulated expression and localization were verified by immunoblot analysis and immunofluorescence microscopy, respectively. Expression of claudin-4 increased TER in both cell lines, whereas effects of the others on TER were variable. Claudin-4 and -11 decreased paracellular permeability for Na+ in MDCK II cells, whereas neither claudin-2 nor -15 had an effect. Conversely, in LLC-PK1 cells, claudin-2 and -15 increased the permeability for Na+, whereas claudin-4 and -11 were without effect. We conclude that the contribution of each claudin is most easily detectable when it reverses the direction of monolayer charge selectivity. These results are consistent with a model in which exogenous claudins add new charge-selective pores, leading to a physiological phenotype that combines endogenous and exogenous contributions. Additionally, it is possible to rationalize the direction of charge selectivity conferred by the individual claudins on the basis of electrostatic effects of the charged amino acids in their first extracellular loops.

An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na(2)HPO(4). 7H(2)O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C.

Sensitization of primary afferent neurons underlies much of the pain and tenderness associated with tissue injury and inflammation. The increase in excitability is caused by chemical agents released at the site of injury. Because recent studies suggest that an increase in voltage-gated Na+ currents may underlie increases in neuronal excitability associated with injury, we have tested the hypothesis that a tetrodotoxin-resistant voltage-gated Na+ current (TTX-R INa), selectively expressed in a subpopulation of sensory neurons with properties of nociceptors, is a target for hyperalgesic agents. Our results indicate that three agents that produce tenderness or hyperalgesia in vivo, prostaglandin E2, adenosine, and serotonin, modulate TTX-R INa. These agents increase the magnitude of the current, shift its conductance-voltage relationship in a hyperpolarized direction, and increase its rate of activation and inactivation. In contrast, thromboxane B2, a cyclooxygenase product that does not produce hyperalgesia, did not affect TTX-R INa. These results suggest that modulation of TTX-R INa is a mechanism for sensitization of mammalian nociceptors.